Resilience of Epiphytic Lichens to Combined Effects of Increasing Nitrogen and Solar Radiation

Fungi Journal of Article Resilience of Epiphytic Lichens to Combined Effects of Increasing Nitrogen and Solar Radiation Lourdes Morillas 1,*, Javier Roales 1,2 , Cristina Cruz 1and Silvana Munzi 1,3   Citation: Morillas, L.; Roales, J.; Cruz, C.; Munzi, S. Resilience of Epiphytic Lichens to Combined Effects of Increasing Nitrogen and Solar Radiation. J. Fungi 2021,7, 333. https://doi.org/10.3390/jof7050333 Academic Editor: Pradeep K. Divakar Received: 27 March 2021 Accepted: 22 April 2021 Published: 26 April 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Centre for Ecology, Evolution and Environmental Changes, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Bloco C2, 1749-016 Lisbon, Portugal; [email protected] (J.R.); [email protected] (C.C.); [email protected] (S.M.) 2 Departamento de Sistemas Físicos, Químicos y Naturales, Universidad Pablo de Olavide, Ctra. Utrera Km 1, 41013 Seville, Spain 3Centro Interuniversitário de História das Ciências e da Tecnologia Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisbon, Portugal *Correspondence: [email protected] Abstract: Lichens are classified into different functional groups depending on their ecological and physiological response to a given environmental stressor. However, knowledge on lichen response to the synergistic effect of multiple environmental factors is extremely scarce, although vital to get a comprehensive understanding of the effects of global change. We exposed six lichen species belonging to different functional groups to the combined effects of two nitrogen (N) doses and direct sunlight involving both high temperatures and ultraviolet (UV) radiation for 58 days. Irrespective of their functional group, all species showed a homogenous response to N with cumulative, detrimental effects and an inability to recover following sunlight, UV exposure. Moreover, solar radiation made a tolerant species more prone to N pollution’s effects. Our results draw attention to the combined effects of global change and other environmental drivers on canopy defoliation and tree death, with consequences for the protection of ecosystems. Keywords: nitrogen pollution; forest decay; global change; chlorophyll fluorescence; Fv/Fm ratio; mediterranean ecosystems 1. Introduction In the last decade, there has been a growing awareness of forests worldwide being affected by severe decline and mortality driven by biotic and abiotic factors, probably a consequence of global change [ 1 – 3 ]. Particularly, in Mediterranean ecosystems located in southern Europe, a severe decline of Quercus suber and Quercus ilex has been detected since the early 1980s [ 4 – 7 ]. In a context where human-induced stressors (such as global warming, air pollutants, and invasive exotic species, among others) are weakening and predisposing trees to defoliation and death, there is a pressing need to understand how these changes can translate into disturbed ecosystem functioning [ 8 ]. Not only that but also how these changes can interact with other environmental stressors at local or intermediate scales, such as atmospheric pollutants. Nitrogen (N) deposition is one of the most challenging drivers of global change in the study area, as N inputs in the Mediterranean Basin are expected to increase from 7 kg N ha −1 yr −1 of the mid-1990s to 12 kg N ha −1 yr −1 in 2050 [ 9 ]. Interactions between environmental changes could trigger complex responses subjected to important uncertainties [ 10 , 11 ], which could be pivotal drivers of the ecosystem dynamics [ 12 ]. However, our understanding of the consequences of forest decline and its interactions with N deposition is constrained by the limited available tools for disentangling these ecological interactions. Epiphytic lichens can have a large impact on forest dynamics (e.g., N, carbon (C), phosphorous (P), and water cycling) [ 13 ], and due to their ability to regulate canopy J. Fungi 2021,7, 333. https://doi.org/10.3390/jof7050333 https://www.mdpi.com/journal/jof J. Fungi 2021,7, 333 2 of 13 environments, can improve water use by plants [ 14 ]. Hence, changes in lichen communities could deeply affect other forest processes. To fully understand the implications of the interactive effect of forest decline and increased N inputs for ecosystem functioning, it becomes fundamental to assess their impacts on epiphytic lichens. A direct consequence of tree defoliation and mortality is the substantial increase in light intensity and, therefore, temperature for epiphytic lichens inhabiting the bark and branches. Such alterations can have a strong effect on lichens, which are amongst the most sensitive organisms to environmental changes [ 15 ], providing meaningful ecological systems to model and foresee the response of other less sensitive organisms of the ecosystem [ 16 , 17 ]. These poikilohydric organisms are especially sensitive to climate [ 15 , 18 ] and air pollution [ 19 – 21 ] because their physiology is tightly linked to their water status, and they lack mechanisms to control their water and nutrient contents [ 22 ]. For this reason, they have been considered as valuable ecological indicators of environmental factors such as temperature and N deposition [15,21,23]. Lichen functional groups are defined as groups of species that respond in a similar way to specific environmental factors [ 24 ]. To attribute a species to a given functional group, researchers assess the data available in the literature (e.g., LIAS [ 25 ]; Nimis and Martellos [ 26 ]; United States Forest Service [ 27 ]), and this knowledge has frequently been used to support environmental policies [ 28 ]. Therefore, the scientific community has considered of pivotal relevance to keep knowledge about species used in monitoring surveys as detailed and updated as possible [ 21 ]. This has been the case for lichens used to identify N pollution status: Oligotrophic species are highly sensitive to N pollution, whereas nitrophytic species tolerate such conditions [ 16 ]. This way, for example, it is currently well documented that Evernia prunastri (L.) and species belonging to the genus Usnea are sensitive species that tend to be affected by increased N availability [ 29 – 32 ], while Xanthoria parietina (L.) is much more resilient to this pollutant [ 16 , 30 , 31 ]. Despite the great efforts to increase our knowledge on the lichen community response to specific environmental factors, there is great uncertainty on how lichens will respond to the synergistic effect of multiple environmental changes. In particular, the combined effect of exposure to solar irradiation and increasing N availability on lichens has never been studied. Similarly, there is very little information about the recovering capacity of lichens after exposure to stress and none about the above-mentioned factors. Filling these gaps is vital to get a comprehensive understanding of the potential effects of global change, not only on lichen communities but also on important ecosystem processes, which is required for the science-based establishment of environmental policies. The aim of this research was to assess the combined effect of both increased solar exposure and increased N deposition on the physiological response of six epiphytic lichen species belonging to various functional groups in terms of solar radiation and N tolerance. We hypothesized that the physiological response of lichens to increased solar radiation would be modulated by the N addition treatment, meaning a lower vitality in N-treated samples exposed to direct sunlight than in control ones. We also expected that the different species would respond differently to these environmental factors because of their adaptation strategies. Namely, we expected that the most tolerant species to each one of these environmental stressors would be the most resilient to their synergistic effect. Finally, we hypothesized that species belonging to more tolerant functional groups would show a higher capacity to recover after the stress exposure than sensitive species. To test our hypotheses, we used chlorophyll afluorescence as a sensitive but non-destructive method for assessing the response of photosynthetic organisms to environmental changes [ 33 ]. Understanding the response of these sensitive elements of ecosystems to multiple environmental stressors is a primary goal to improve the use of lichens as indicators for environmental protection and predict the potential consequences of global change on ecosystem functioning. J. Fungi 2021,7, 333 3 of 13 2. Materials and Methods 2.1. Lichen Sampling In spring 2020, samples of Xanthoria parietina,Ramalina lacera,Usnea sp., Flavoparmelia caperata,Parmotrema hypoleucinum, and Evernia prunastri were collected from a forest patch near Vila Franca de Xira, Portugal (191 m a.s.L., 38 ◦ 58 0 29 00 N, 9 ◦ 00 0 27 00 W). This site was devoid of important sources of pollution; however, it can receive low N deposition from local roads and agricultural areas. Whenever possible, lichens were collected whole by cutting the branch or removing the bark that acted as a substrate. Samples were then transported to the laboratory, where branches or bark were carefully cut in order to leave only the minimum necessary to support the lichen. Finally, lichens were carefully cleaned to remove any impurities. All lichens were stored at room temperature and were fully rehydrated before performing any measurements. The Ecological Indicator Values (Table 1) established by Nimis and Martellos [ 26 ] were used in this study as a proxy of functional groups. This table shows categorical values ranging from 1 to 5 that represent the ecological situations regarding solar irradiation and eutrophication where these species can be found in nature. Although samples of Usnea were not identified to species, most species of this genus have very similar ecological requirements with high tolerance to solar radiation and high sensitivity to N. Table 1. Lichen’s Ecological Indicator Values established by Nimis and Martellos (2020). Lichen Species Solar Irradiation 1Eutrophication 2 Xanthoria parietina 3, 4, 5 3, 4 Ramalina lacera 4, 5 2, 3 Usnea sp. 4, 5 1, 2 Flavoparmelia caperata 3, 4 1, 2, 3 Parmotrema hypoleucinum 4, 5 1, 2 Evernia prunastri 3, 4, 5 1, 2, 3 1 Values mean: 1 in very shaded situations, 2 in shaded situations, 3 in sites with plenty of diffuse light but scarce direct solar irradiation, 4 in sun-exposed sites, 5 in sites with very high direct solar irradiation. 2 Values mean: 1 no eutrophication , 2 very weak eutrophication, 3 weak eutrophication, 4 rather high eutrophication, 5 very high eutrophication. 2.2. Nitrogen Treatment Two levels of N treatment (N 25 and N 50) and a control were established. Treatments involved daily immersion of the samples in 25 mM (N 25) or 50 mM (N 50) (NH 4 ) 2 SO 4 solutions for 5 min repeated for 58 days. The same protocol was followed for control samples using only mineral water with low mineral content. It is worth noting that all species in this experiment have a trebouxioid photobiont, which requires alternate wetting and drying to get a photosynthetic response of the photobiont. Lichens were housed in custom-built wire mesh cages that prevented their flotation on the solutions (see Figure S1 in the Supplementary Materials ). After each treatment, the samples were placed in wire mesh shelves in a well-aerated location, which ensured enough drying before the subsequent immersion and hence prevented rotting of the lichen thalli or the substrate (see Figure S2 in the Supplementary Materials). Mineral water with low mineral content was used to avoid osmotic shock. For each treatment, 5 replicates were used. 2.3. Increased Radiation Treatment Light exposure treatment was undertaken in 3 stages in parallel with the N treatment. First, lichens were incubated indoors in front of a big north-facing window, with abundant light but no direct sun. This started the same day as the N treatment and lasted 20 days. The 2nd stage involved the daily exposure of the lichens to 5 h of direct sunlight during the hours of higher solar radiation (from 10 a.m. to 3 p.m., solar time). The average temperature during the exposure was 28 ◦ C, and the maximum temperature was 35.8 ◦ C, with sunny conditions throughout the exposure. The reported temperature was measured in the closest meteorological station, therefore, in shaded conditions. It should be noted that J. Fungi 2021,7, 333 4 of 13 the temperature at the lichen surface, which was permanently facing the sun, was likely to exceed the measured air temperature considerably. This part of the treatment lasted 8 days and was performed in mid-May in southern Spain (42 m a.s.l., 37 ◦ 16 0 51 00 N, 5 ◦ 54 0 54 00 W). After each sun exposure, lichens were returned indoors to avoid humidity or temperature fluctuations. In the last stage, intended as recovery, lichens were returned to the indoors location. The recovery lasted 29 days until the end of the experiment. No control group was established for this treatment because we intended to analyze the difference among N levels and not radiation levels. 2.4. Chlorophyll a Fluorescence Treatment effects were quantified using a Plant Efficiency Analyzer Handy PEA (Hansatech Instruments LTD, Pentney, UK), which determined the Fv/Fm ratio, the most frequently used chlorophyll afluorescence parameter, vitality index in ecological research [ 34 – 36 ]. Ten minutes after each daily N treatment, with the samples fully rehydrated, the Fv/Fm ratio was measured [ 37 , 38 ]. Lichens were dark-adapted at room temperature for 15 min to maximize oxidation of the primary quinone electron acceptor of PSII immediately before measuring fluorescence. For each treatment, 5 replicates were used. 2.5. Statistical Analyses Data were checked for conformity with repeated measures ANOVA assumptions via Shapiro–Wilk normality test and Mauchly’s test of sphericity. Normality tests of the residuals by time point revealed that they followed approximately a normal distribution. Sphericity could not be assured due to the number of samples being lower than the number of repeated measures, hence a Greenhouse–Geisser correction was used. Differences in Fv/Fm values among N treated and control samples were evaluated via repeated measures procedure following a 2-way mixed ANOVA design with 1 within-subjects factor and 1 between-groups factor in IBM SPSS Statistics 23.0 (SPSS Inc., Chicago, IL, USA). Pairwise comparisons were performed by comparing main effects through post hoc tests using the Bonferroni correction. To investigate interactions, data were divided into subsets based on N treatments and then were subjected to repeated-measures analyses. The evolution following the recovery period was computed as the difference between Fv/Fm values at day 58 (final time) and Fv/Fm values at day 29 (end of the second stage of the radiation treatment). To test for differences in the ability to recover from the radiation treatment of the different N treatments, we performed 1-way ANOVA of the differences between Fv/Fm values for all the studied epiphytic lichens. 3. Results 3.1. Analyses of the Effect of N Pollution Six measurements over the first 20 days assessed the effects of N pollution alone, while lichens were not subject to irradiation. Significant effects of the different N treatments and time for all species except Xanthoria were found (Figure 1and Table 2). For all affected species, Fv/Fm values gradually dropped, responding to the cumulative effect of N, especially the 50 N dose (Figure 1). In Evernia and Flavoparmelia there was no significant effect of the low N dose but a significant reduction with the high N dose compared with both the control and low N dose. In Parmotrema,Ramalina and Usnea, both N doses caused significant reductions, but the level of reduction between the N doses was statistically indistinguishable. Measurements of the control indicated there was no deterioration in the index due to time, i.e., the experimental conditions did not affect the measurements. However, with the N treatments, as the experimental duration progressed, the lichens received more N, i.e., time also means a greater cumulative N dose. We detected significant differences between the control and 50 N and 25 N and 50 N but not between control and 25 N for Evernia ( pcontrol-25 N = 0.960 , pcontrol-50 N < 0.0005 , p25 N–50 N < 0.0005 ) and Flavoparmelia ( pcontrol-25 N = 0.534 ,p control-50 N = 0.001, p25 N–50 N = 0.009 ). These differences were also found among control and N-treated samples, both 25 and 50 N, although not between J. Fungi 2021,7, 333 5 of 13 them, for Parmotrema (p control-25 N = 0.002, pcontrol-50 N = 0.002 , p25 N–50 N = 1.000 ), Ramalina ( pcontrol-25 N < 0.0005 ,p control-50 N < 0.0001, p25 N–50 N = 1.000 ) andUsnea ( pcontrol-25 N < 0.0005 , p control-50 N < 0.0005, p 25 N–50 N = 1.000). There was a significant N treatment × time interaction for Ramalina,Flavoparmelia,Usnea, and Evernia, and when looking into them, we found a significant effect of time for 25 N and 50 N treatments, but not for control for Ramalina, Usnea, and Evernia (Table 2). In the case of Flavoparmelia, we only detected a significant effect of time for 50 N (Table 2). J. Fungi 2021, 7, x FOR PEER REVIEW 5 of 14 received more N, i.e., time also means a greater cumulative N dose. We detected significant differences between the control and 50 N and 25 N and 50 N but not between control and 25 N for Evernia (p control-25 N = 0.960, p control-50 N < 0.0005, p 25 N–50 N < 0.0005) and Flavoparmelia (p control-25 N = 0.534, p control-50 N = 0.001, p 25 N–50 N = 0.009). These differences were also found among control and N-treated samples, both 25 and 50 N, although not between them, for Parmotrema (p control-25 N = 0.002, p control-50 N = 0.002, p 25 N–50 N = 1.000), Ramalina (p control-25 N < 0.0005, p control-50 N < 0.0001, p 25 N–50 N = 1.000) and Usnea (p control-25 N < 0.0005, p control50 N < 0.0005, p 25 N–50 N = 1.000). There was a significant N treatment × time interaction for Ramalina, Flavoparmelia, Usnea, and Evernia, and when looking into them, we found a significant effect of time for 25 N and 50 N treatments, but not for control for Ramalina, Usnea, and Evernia (Table 2). In the case of Flavoparmelia, we only detected a significant effect of time for 50 N (Table 2). Figure 1. Temporal evolution of Fv/Fm ratio during 58 days for Control (no nitrogen [N] addition), 25 N (treated with 25 mM of (NH4)2SO4) and 50 N (treated with 50 mM of (NH4)2SO4) for the following species: (a) Xanthoria parietina, (b) Ramalina lacera, (c) Usnea sp., (d) Flavoparmelia caperata, (e) Parmotrema hypoleucinum, (f) Evernia prunastri. All samples were subjected to the same radiation treatment. The period of exposure to direct sunlight is indicated in yellow. Lowercase letters indicate significant differences among N treatments for each species (n = 5). Table 2. Repeated measures ANOVA analyses for all the studied epiphytic lichens during the first 20 days. The samples were subjected to N treatment alone during this period. Control = No nitrogen (N) addition, 25 N = treated with 25 mM of (NH4)2SO4, 50 N = treated with 50 mM of (NH4)2SO4. Lichen Species Factor df F p Xanthoria parietina N treatment 2 0.198 0.823 Time 2.870 1.912 0.148 N treatment × Time 5.740 0.617 0.709 Ramalina lacera N treatment 2 23.679 <0.0005 Figure 1. Temporal evolution of Fv/Fm ratio during 58 days for Control (no nitrogen [N] addition), 25 N (treated with 25 mM of (NH 4 ) 2 SO 4 ) and 50 N (treated with 50 mM of (NH 4 ) 2 SO 4 ) for the following species: ( a )Xanthoria parietina, (b)Ramalina lacera , ( c )Usnea sp., ( d )Flavoparmelia caperata, ( e )Parmotrema hypoleucinum, ( f )Evernia prunastri. All samples were subjected to the same radiation treatment. The period of exposure to direct sunlight is indicated in yellow. Lowercase letters indicate significant differences among N treatments for each species (n= 5). Table 2. Repeated measures ANOVA analyses for all the studied epiphytic lichens during the first 20 days . The samples were subjected to N treatment alone during this period. Control = No nitrogen (N) addition, 25 N = treated with 25 mM of (NH4)2SO4, 50 N = treated with 50 mM of (NH4)2SO4. Lichen Species Factor df F p Xanthoria parietina N treatment 2 0.198 0.823 Time 2.870 1.912 0.148 N treatment ×Time 5.740 0.617 0.709 Ramalina lacera N treatment 2 23.679 <0.0005 Time 2.707 20.028 <0.0005 N treatment ×Time 5.413 3.857 0.006 J. Fungi 2021,7, 333 6 of 13 Table 2. Cont. Lichen Species Factor df F p Interaction N treatment ×Time Control 1.999 0.920 0.437 Interaction N treatment ×Time 25 N 1.369 17.619 0.005 Interaction N treatment ×Time 50 N 2.247 9.150 0.006 Usnea sp. N treatment 2 22.354 <0.0005 Time 2.418 19.147 <0.0005 N treatment ×Time 4.836 3.428 0.016 Interaction N treatment ×Time Control 2.102 3.194 0.091 Interaction N treatment ×Time 25 N 2.752 15.265 <0.0005 Interaction N treatment ×Time 50 N 1.458 6.499 0.038 Flavoparmelia caperata N treatment 2 14.098 0.001 Time 2.310 7.787 0.001 N treatment ×Time 4.620 4.170 0.007 Interaction N treatment ×Time Control 2.612 1.626 0.243 Interaction N treatment ×Time 25 N 2.133 3.792 0.064 Interaction N treatment ×Time 50 N 2.060 5.772 0.027 Parmotrema hypoleucinum N treatment 2 14.291 0.001 Time 3.032 5.077 0.005 N treatment ×Time 6.064 1.531 0.195 Evernia prunastri N treatment 2 34.371 <0.0005 Time 3.066 28.366 <0.0005 N treatment ×Time 6.132 5.988 <0.0005 Interaction N treatment ×Time Control 1.911 3.591 0.081 Interaction N treatment ×Time 25 N 2.283 6.503 0.024 Interaction N treatment ×Time 50 N 1.877 21.993 0.001 3.2. Analyses of the Synergetic Effect of N and Solar Radiation We found a significant effect of the different N treatments on all lichen species when analyzing the synergetic effect of N and increased solar radiation (Figure 1and Table 3). For all species, direct sun exposure dramatically decreased Fv/Fm (Figure 1), although these values reached their minimum earliest in N treated samples. Lichens exposed to 50 N treatment showed a consistent trend towards lower Fv/Fm values for all species (Figure 1) . We detected significant differences between the control and 50 N and 25 N and 50 N but not between control and 25 N for Xanthoria (p control-25 N = 1.000, pcontrol-50 N = 0.003 , p25 N–50 N = 0.009) and Evernia (p control-25 N = 0.133, p control-50 N < 0.0005, p 25 N-50 N = 0.009). Differences were also found among control and N-treated samples, both 25 and 50 N, although not between them, for Parmotrema (p control-25 N < 0.0005, p control-50 N < 0.0005, p25 N–50 N = 0.072 ), Ramalina ( pcontrol-25 N < 0.0005, p control-50 N < 0.0005, p 25 N–50 N = 0.282) and Usnea ( pcontrol-25 N < 0.0005 ,p control-50 N < 0.0005, p 25 N–50 N = 1.000). We found significant differences among all the three groups (control, 25 N and 50 N) for Flavoparmelia ( pcontrol-25 N < 0.0005, p control-50 N < 0.0005, p 25 N–50 N < 0.0005). Time was found to significantly affect all analyzed lichens species (Figure 1and Table 3). There was a significant N treatment × time interaction for Ramalina,Flavoparmelia, and Evernia, and when looking into them, we found a significant effect of time for the control, 25 N and 50 N treatments for all these species (Table 3). J. Fungi 2021,7, 333 7 of 13 Table 3. Repeated measures ANOVA analyses for all the studied epiphytic lichens during the whole experiment. The samples were subjected to N treatment and solar radiation. Control = No nitrogen (N) addition, 25 N = treated with 25 mM of (NH4)2SO4, 50 N = treated with 50 mM of (NH4)2SO4. Lichen Species Factor df F p Xanthoria parietina N treatment 2 10.769 0.002 Time 4.753 71.389 <0.0005 N treatment ×Time 9.505 1.230 0.293 Ramalina lacera N treatment 2 112.702 <0.0005 Time 5.020 85.423 <0.0005 N treatment ×Time 10.039 2.083 0.040 Interaction N treatment ×Time Control 3.381 12.826 <0.0005 Interaction N treatment ×Time 25 N 2.000 41.841 <0.0005 Interaction N treatment ×Time 50 N 2.786 80.237 <0.0005 Usnea sp. N treatment 2 99.448 <0.0005 Time 4.560 137.635 <0.0005 N treatment ×Time 9.119 1.527 0.164 Flavoparmelia caperata N treatment 2 372.020 <0.0005 Time 5.163 36.500 <0.0005 N treatment ×Time 10.326 4.744 <0.0005 Interaction N treatment ×Time Control 2.369 4.875 0.031 Interaction N treatment ×Time 25 N 2.545 13.058 0.001 Interaction N treatment ×Time 50 N 2.425 37.915 <0.0005 Parmotrema hypoleucinum N treatment 2 46.314 <0.0005 Time 6.299 32.540 <0.0005 N treatment ×Time 12.598 1.629 0.098 Evernia prunastri N treatment 2 21.052 <0.0005 Time 6.129 135.430 <0.0005 N treatment ×Time 12.258 7.621 <0.0005 Interaction N treatment ×Time Control 2.517 86.264 <0.0005 Interaction N treatment ×Time 25 N 2.663 59.673 <0.0005 Interaction N treatment ×Time 50 N 3.231 25.576 <0.0005 3.3. Analyses of the Evolution Following the Recovery Period None of the studied species was able to recover following the solar radiation exposure (Figures 1and 2). However, a non-significant trend of decreased ability to recover from solar radiation stress with increased N dose was found for Xanthoria,Usnea, and Flavoparmelia (Figure 2and Table 4). J. Fungi 2021,7, 333 8 of 13 J. Fungi 2021, 7, x FOR PEER REVIEW 8 of 14 Figure 2. Recovery capacity for all studied lichens computed as the difference between Fv/Fm ratio at the end of the experiment (day 58) and Fv/Fm ratio at the end of the direct sunlight exposure (day 29) for the control (no nitrogen [N] addition), 25 N (treated with 25 mM of (NH4)2SO4), and 50 N (treated with 50 mM of (NH4)2SO4). No significant differences among N treatments were found for any species (n = 5). Table 4. One-way ANOVA of the Fv/Fm evolution following the recovery period for all the studied epiphytic lichens. Lichen Species df F p Xanthoria parietina 2 0.876 0.442 Ramalina lacera 2 0.304 0.743 Usnea sp. 2 0.280 0.761 Flavoparmelia caperata 2 2.379 0.135 Parmotrema hypoleucinum 2 0.445 0.651 Evernia prunastri 2 0.789 0.477 4. Discussion In general, our results showed a detrimental cumulative effect of N and solar radiation exposure on lichens but did not support the view that increased N availability constrained the physiological ability of lichens to cope with increased sunlight exposure, disproving our first hypothesis. However, in the case of Xanthoria, we found a differential response: It was unaffected by N addition, but solar exposure promoted an N dose-dependent response of the Fv/Fm parameter. Therefore, the physiological response of this species to increased N addition was modulated by solar radiation. Our second hypothesis, which states that different lichen species would vary in their response to the synergetic effect of increased N doses and solar exposure, was not supported by our data; we expected that the most tolerant species to each one of these environmental stressors, i.e., Xanthoria, would be the most resilient to their synergistic effect. In contrast to our expectations, we only found slight differences among species, as at the end of the combined treatment, Xanthoria was affected to the same extent as the rest of the analyzed species. Similarly, our last hypothesis was not supported because more tolerant functional groups were equally unable to recover after sunlight exposure as sensitive species. These results indicate that a reduction of vitality among the epiphytic community should be expected in the face of global change. Increased N deposition that is projected to occur in the next decades in Mediterranean regions [9] has previously been pinpointed as a key factor underpinning reduced vitality in many epiphytic lichens [39]. Our data during 20 days of N addition, pre solar radiation, support this view, with reduced vitality in N sensitive species. Other researchers have found a fertilizing effect of moderate N Figure 2. Recovery capacity for all studied lichens computed as the difference between Fv/Fm ratio at the end of the experiment (day 58) and Fv/Fm ratio at the end of the direct sunlight exposure ( day 29 ) for the control (no nitrogen [N] addition), 25 N (treated with 25 mM of (NH 4 ) 2 SO 4 ), and 50 N (treated with 50 mM of (NH 4 ) 2 SO 4 ). No significant differences among N treatments were found for any species (n= 5). Table 4. One-way ANOVA of the Fv/Fm evolution following the recovery period for all the studied epiphytic lichens. Lichen Species df F p Xanthoria parietina 2 0.876 0.442 Ramalina lacera 2 0.304 0.743 Usnea sp. 2 0.280 0.761 Flavoparmelia caperata 2 2.379 0.135 Parmotrema hypoleucinum 2 0.445 0.651 Evernia prunastri 2 0.789 0.477 4. Discussion In general, our results showed a detrimental cumulative effect of N and solar radiation exposure on lichens but did not support the view that increased N availability constrained the physiological ability of lichens to cope with increased sunlight exposure, disproving our first hypothesis. However, in the case of Xanthoria, we found a differential response: It was unaffected by N addition, but solar exposure promoted an N dose-dependent response of the Fv/Fm parameter. Therefore, the physiological response of this species to increased N addition was modulated by solar radiation. Our second hypothesis, which states that different lichen species would vary in their response to the synergetic effect of increased N doses and solar exposure, was not supported by our data; we expected that the most tolerant species to each one of these environmental stressors, i.e., Xanthoria, would be the most resilient to their synergistic effect. In contrast to our expectations, we only found slight differences among species, as at the end of the combined treatment, Xanthoria was affected to the same extent as the rest of the analyzed species. Similarly, our last hypothesis was not supported because more tolerant functional groups were equally unable to recover after sunlight exposure as sensitive species. These results indicate that a reduction of vitality among the epiphytic community should be expected in the face of global change. Increased N deposition that is projected to occur in the next decades in Mediterranean regions [ 9 ] has previously been pinpointed as a key factor underpinning reduced vitality in many epiphytic lichens [ 39 ]. Our data during 20 days of N addition, pre solar radiation, support this view, with reduced vitality in N sensitive species. Other researchers have found a fertilizing effect of moderate N supply J. Fungi 2021,7, 333 9 of 13 shown by increased thalli N, chlorophyll concentration, and rates of photosynthesis [ 21 , 40 ]. It is well documented that N performs as a nutrient below the toxicity threshold, especially in oligotrophic environments [ 41 – 43 ]. However, we did not observe this, possibly because the lichens came from a site with limited N contribution from surrounding roads and agricultural areas. Thus, even modest N addition doses during our experiment exceeded the fertilization threshold and performed as a pollutant. Direct exposure to high solar radiation can also cause stress and threaten epiphytic lichens. Some lichens appear to be highly sensitive to sudden solar exposure increase [ 44 ], such as that observed in anthropogenically induced forest decline. This is despite the varied protective mechanisms lichens employ to avoid damage from high insolation such as colored cortical secondary compounds (e.g., usnic acid, parietin, and melanic compounds, [ 45 , 46 ]), thick cortical layer [ 47 – 49 ], hairiness [ 50 ], light-reflecting calcium oxalate crystals, or biochemical protective mechanisms [ 51 ]. A number of studies report a high natural radiation susceptibility of photosystem II in lichens [ 44 , 52 ], with particular sensitivity to both excessive ultraviolet (UV) [ 53 ] and photosynthetically active radiation [ 54 ]. Likewise, the increased temperature has also been highlighted as a major damaging factor for epiphytic lichen vitality. Smith and collaborators [ 55 ] found that warm climate tolerant lichen communities are already close to exceeding their upper climatic limits and are even more vulnerable to increased temperatures than high-elevation lichens. Our experimental design, which accounted for exposing wet lichens to direct solar radiation, does not allow us to discriminate between the effect of increased radiation and temperature. However, when Gauslaa and Solhaug [ 56 ] enclosed thalli of various lichens in a desiccator at 15 ◦ C illuminated at 100 mol m −2 s −1 , periodic measurements of Fv/Fm showed a decreasing trend. They then removed all uppermost thalli exposed to the irradiance and measured thalli that had been shaded by the previously harvested thalli, finding that Fv/Fm values suddenly rose close to pre-desiccation levels (Gauslaa and Solhaug, unpublished data). Based on these findings, it is reasonable to think that the damaging factor operating in our experiment could be increased radiation more than temperature, although the interactive effect of both factors is also a plausible possibility. Accordingly, Gauslaa and Solhaug [ 57 ] reported a photoinhibition effect on lichen thalli due to heat and light stress both separately and combined. Hydration status is a key determinant in how lichens respond to radiation. Wet thalli are more susceptible to irradiance than dried thalli because humidity increases the translucency of the protective upper cortex, increasing light absorbance [ 58 , 59 ]. Therefore, photosystem II needs more protection against the harmful excess light energy compared to desiccated thalli [ 60 ], in which case most of the radiation is reflected from the thalli surface [ 58 ]. Besides this generic aspect that might have had a role in the striking and undivided negative response of all studied lichens to solar radiation, hydration appears to deeply affect photoinhibition in a species-specific manner. Looking at the control thalli, the least affected by solar radiation was F. caperata. This could appear in contradiction with the fact that F. caperata is the only one among the species used in the experiment, adapted to diffuse light more than to direct solar radiation [ 26 ]. However, shade-adapted species were found to be most affected by radiation when they are dry, whereas sun-adapted species tend to be more susceptible when they are wet [ 56 ]. Tretiach et al. [ 61 ] showed that when hydrated, F. caperata activate repair mechanisms like ROS-scavenging enzymes and oxidation of polyols and phenols that allow it to survive in case of environmental stress. Since our samples were fully hydrated every day, we can reasonably argue that thalli could restore a proper enzymatic activity and antioxidants concentration daily [ 62 ]. In agreement, Gauslaa and Solhaug [ 56 ] observed that the sun-adapted X. parietina was less affected by radiation in its desiccated state, whereas hydration caused increased photoinhibition. These authors concluded that the extent of solar damage varies more among species when dry lichens are exposed to radiation than when radiation affects wet lichens. This, and the fact that all tested lichens shared a trebouxioid photobiont fully active following alternate wetting and drying cycles, could explain why we found such a homogeneous response to